【作者】 寸艳萍；

【导师】 胡志安；

【作者基本信息】 第三军医大学 ， 生理学， 2010， 硕士

【摘要】 Orexin[又称hypocretin)是在外侧下丘脑区(lateral hypothalamic area, LHA)合成的具有神经兴奋作用的小分子肽。Orexin有两个单体：orexin A和orexin B,它们来自同一基因转录产物—前orexin原的酶解产物,主要通过两个G蛋白偶联受体orexin-1受体(orexin-1 receptor,OX1R)和orexin-2受体(orexin-2 receptor,OX2R)发挥作用。Orexin神经元胞体位于下丘脑侧下及穹窿周围区核团,数量仅约数千个但其纤维及受体分布却十分广泛,又有明显的投射投向多个脑区。中枢orexin系统对觉醒起关键作用：orexin能神经元密集地投向参与睡眠调控的脑区;orexin基因敲除小鼠或其受体基因自发突变后导致发作性睡病样表型包括猝倒和病态快速动眼(rapid eye movement, REM)睡眠。另外,人发作性睡病患者脑脊液及血浆中orexin水平也有降低。LHA的orexin神经元不仅作为觉醒激动系统中的一个结构参与觉醒促进作用,而且还通过对其他觉醒启动系统的统一控制实现睡眠-觉醒的调控。研究发现,在LHA微注射orexin A能够增加Glu释放,并且在LHA注射Glu促进觉醒。尽管如此,与Glu突触传递有关的orexin能神经元激活对睡眠-觉醒的调节机制尚未阐明。腺苷(adenosine, AD)是广泛存在于中枢神经系统细胞内、外的一种小分子物质,属于中枢抑制性递质,具有强烈抑制胆碱能和谷氨酸能神经元的功能。在中枢神经系统有4个G蛋白偶联的AD受体A1R,A2aR,A2bR和A3R,其中A1R,A2aR与睡眠-觉醒状态的调节有关。传统观点认为AD的作用位点主要位于基底前脑,但是最近—项下丘脑脑片的膜片钳实验证明,在外侧下丘脑这个觉醒促进区给外源性AD后,能抑制orexin能神经元的活性。本课题组前期采用脑片膜片钳技术研究发现,内源性AD通过激活其A1R抑制orexin能神经元的兴奋性突触传入。AD为生理性睡眠因子,在外侧下丘脑给予AD受体拟似剂(N6-cyclopentyladenosine, CPA)可诱发睡眠,而给予AD受体选择性的拮抗剂(1,3-dipropyl-8-phenylxanthine, DPX)可以促进觉醒,提示A1R主要参与其中。这些证据表明外侧下丘脑是AD调节睡眠-觉醒周期的另一个重要靶区。尽管如此,A2R是否参与外侧下丘脑区AD对睡眠-觉醒状态的调节以及在体情况下orexin对睡眠稳态因子AD催眠效应的调节尚未有文献报道。本研究采用多导睡眠记录和微注射技术,首先观察与谷氨酸能突触传递有关的orexin能神经元激活对睡眠-觉醒的影响,然后明确AD及其受体对外侧下丘脑orexin能神经元的抑制作用及orexin对睡眠稳态因子AD催眠效应的调节作用。结果如下：1. orexin A对睡眠-觉醒的调节作用外侧下丘脑orexin能神经元区内微量注射orexin A对大鼠睡眠-觉醒周期的影响。在LHA微量注射后的0-3 h内,给予10 pmol orexin A的大鼠觉醒期显著增加到微注射ACSF后的1.28倍,基线对照的1.31倍(P<0.05)；非快速眼动(non-rapid eye movement, NREM)睡眠显著缩短到微注射ACSF后的90%,基线对照的89%(P<0.05)；此时REM睡眠没有明显差异。在给予40 pmol orexin A后的0-3 h内,大鼠的觉醒期显著增加到微注射ACSF后的1.32倍,基线对照的1.35倍,并且NREM睡眠和REM睡眠均显著缩短(P<0.05)。在注射orexin A之前预先在相同位点微量注射SB-334867(OX1R特异性阻断剂)后,orexin A的促觉醒效应被明显抑制,表明OXlR介导了orexin A的作用。2.局部谷氨酸能神经元环路影响orexin A的促觉醒效应与ACSF自身对照相比,在LHA单侧微量注射80 ng Glu后,大鼠的觉醒时间在0～3h内增加到1.39倍,并且NREM睡眠时间减少到79%。而单侧微量注射40 ng Glu引起的睡眠-觉醒变化没有统计学差异。微量注射ACSF/orexin A后,0-3 h内的觉醒时间增加ACSF自身对照的1.36倍,而在给予orexin A之前预先注射Glu NMDA受体拮抗剂D-AP5后,orexin A的促觉醒效应被抑制,大鼠的睡眠-觉醒时间回复到ACSF自身对照水平。3.AD对睡眠-觉醒的调节作用在外侧下丘脑微注射AD 1、10 nmol和20 nmol后的0-3 h内大鼠觉醒时间分别减少到ACSF自身对照的84%、62%和60%；并且在10 nmol和20 nmol的高剂量AD给药后还伴有NREM和REM睡眠的显著增加。在3-6 h内SD大鼠ACSF自身对照和AD给药后的觉醒时间、NREM和REM睡眠时间在统计学上没有显著性差异。与ACSF自身对照相比,在给大鼠外侧下丘脑单侧微注射AD 20 nmol的第1、第2和第3 h内,AD明显减少觉醒时间(P<0.05),分别减少到ACSF自身对照的63%、61%和53%。并且在AD给药后的第5 h大鼠觉醒时间明显降低至ACSF自身对照的78%。此外给予A1R拮抗剂DPX促进大鼠觉醒,而给予A2R拮抗剂3,7-dimethyl-1-propargylxanthine (DMPX)没有类似效应,提示AD通过激活A1R,而不是A2R,发挥促睡眠效应。4. orexin A对AD促睡眠效应的抑制作用与自身对照组比较,在LHA微量注射AD/ACSF后的0-3 h内,大鼠觉醒时间减少到74%,并且NREM和REM睡眠分别增加到1.10、1.40倍(P<0.05)。当微注射AD/orexin A后,觉醒时间、NREM睡眠时间和REM睡眠时间回复到原来水平,与自身对照日没有显著的差异(P>0.05)。在微量注射后的3-6 h内,与自身对照组比较,AD/ACSF和AD/orexin A的觉醒、NREM和REM睡眠的变化差异无统计学意义(P>0.05)。此外,通过比较AD/ACSF和AD/orexin A的睡眠-觉醒调节作用,提示orexin A抑制AD的促睡眠作用。综上所述,本研究结果表明：第一,LHA微注射orexin A促进大鼠觉醒,并且减少NREM和REM睡眠。第二,外侧下丘脑orexin A通过局部谷氨酸能神经元环路发挥其促觉醒效应。第三,LHA微注射AD通过激活A1R,而不是A2R发挥促睡眠效应,并且orexin A能够拮抗其催眠效应。更多还原

【Abstract】 The neuropeptide orexins, also called hypocretins, are produced by orexinergic neurons in the lateral hypothalamus area (LHA). Orexin system includes two separate peptides orexin A and B proteolytically derived from the same precursor protein and two specific G-protein-coupled receptors OX1R and OX2R.Although orexin neurons are localized in a subregion of hypothalamus, their projections and orexin receptors are widely distributed in the brain. The central orexin system plays a critical role in regulating sleep-wake cycle:the orexinergic neurons have extensive projections to the arousal systems; the loss of orexinergic cells or its malfunction has been linked to cataplexy like state in mice and abnormal rapid eye movement (REM) sleep. The orexin levels are low in cerebrospinal fluid and blood plasm. Orexins and orexin receptors play highly important roles in regulating sleep-awake states and the maintenance of arousal by regulating monoaminergic/cholinergic nuclei in the brain. The microinjection of orexin A into the lateral hypothalamus increase glutamate release, while glutamic acid stimulation of the lateral hypothalamic area promotes arousal and inhibits NREM/REM sleep. However, the mechanism of glutamatergic activation of orexin neurons on sleep-wake behavioral modulation is not known.Adenosine (AD), a byproduct of cell energy metabolism, serves as an effective sleep-promoting neurotransmitter in the central nervous system (CNS). Four subtypes of G protein-coupled adenosine receptors, A1R, A2aR, A2bR and A3R, are indicated in CNS, while A1Rs and A2aRs are involved in sleep-wake modulation. It is well illustrated that AD increases sleep by inhibition of the cholinergic region in basal forebrain through A1Rs. An in vitro study using electrophysiological methods reports that AD inhibits the activity of orexinergic neurons via A1Rs. A recent study in our lab has shown that AD inhibits the glutamatergic synaptic transmission to orexin neurons via A1Rs. Moreover, administration of adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in the orexinergic LHA could induce sleep and blockade of A1R by application of a selective AD A1R antagonist 1,3-dipropyl-8-phenylxanthine(DPX) could produce a significant increase in wakefulness. These evidences illuminate that there is another important target involved the modulation of endogenous adenosine on sleep-wakefulness in the orexinergic zone of LHA. But until now, whether A2R is involves in the sleep-inducing effect of adenosine in the orexinergic LHA and whether the sleep-inducing effect of adenosine could be abolished by the orexins are still little known.In the present study, using polysomnogram (PSG) recordings and microinjection technique, we firstly observed this modulation of orexinergic neurons which involved in glutamatergic synaptic transmission on spontaneous sleep-wakefulness. Subsequently, we evaluate the inhibition of adenosine and adenosine receptors on the orexin neurons in the lateral hypothamus as well as the excitation of orexin on the sleep-inducing effect of adenosine. The results show as follow:1. The arousal effects of orexin AMicroinjection of 10 pmol orexin A into LHA could increase the total amount of wakefulness to 1.28 and 1.31-fold and decrease the entire time of NREM sleep to 0.90 and 0.89-fold during the first 3-h period, when compared with that of the ACSF treatment and baseline respectively. The change of REM sleep was under statistical level. Similarly, application of 40pmol orexin A into LHA significantly increased the total amount of wakefulness to 1.32 and 1.35-fold during the first 3-h session compared with the ACSF and baseline day respectively. The increase in wakefulness induced by 40 pmol orexin A was concomitant with the reduction in NREM/REM sleep。The time of wakefulness was increased after DMSO/orexin A application during the first 3-h post-injection sessions compared with control, and then recovered approximately to control level after SB-334867/orexin-A treatment.2. Orexin A promotes wakefulness via local glutamatergic neuronsThe significant increase in wakefulness to 1.39-fold (P<0.05) was concomitant with the decrease in NREM sleep to 79%(P<0.05) after the high dose of 80 ng glutamate microinjection during the 3-h post-injection recording period, when compared with ACSF treatment group. There is no significant change in any behavioral state after 40 ng glutamate administation. The ACSF/orexin A administration significantly increased the total amount of wakefulness during the 3-h sessions to 1.36-fold when compared with control, and then reduced approximately to control level after NMD A antagonist D-AP5/orexin A treatment.3. The sleep-inducing effect of ADAD administration to LHA at doses of 1,10,20 nmol decreased the total amount of wakefulness to 84%,62% and 60% during that first 3-h recording period and concomitantly increased NREM and REM sleep, when compared with baseline. AD administration into the LHA at 20 nmol dose shortened the wake time to 63%,61%,53% and 78% at the 1-h, 2-h,3-h and 5-h, respectively, over the ACSF control group. The reduction of wakefulness was concomitant with enhancements in NREM and REM. Microinjection of a selective AD A1R antagonist DPX into LHA could increase wakefulness and decrease sleep, but there is on similar effect on A2R antagonist DMPX application, suggesting A1R but not A2R involves in the sleep-inducing effect of AD.4. Orexin A attenuates the sleep-inducing effect of ADThe total amount of wakefulness after AD/ACSF microinjection was significantly decreased to 74%(P<0.05) during the first 3-h post-injection sessions compared with the baseline, and then recovered approximately to baseline level after AD/orexin A treatment. Accordingly, the NREM and REM sleep was respectively increased to 1.10 and 1.40-fold by microinjection of AD, when compared with baseline, and then both recovered approximately to baseline level after AD/orexin A treatment. Moreover, by comparing the sleep-wake regulation of AD/ACSF and AD/orexin A, we find orexin A inhibits the sleep-inducing effect of AD.In conclusion, these observations provide evidences that the orexin A could promote wake and concomitantly decrease NREM and REM sleep via local glutamate circuits. In addition, AD reduces the amount of wakefulness through A1Rs, but not A2Rs. Furthermore, the sleep-inducing effect of adenosine on sleep-wakefulness is attenuated when orexin A tone was increased.更多还原